II. PROGRESS: Science and Experiment Verification Tests. The major focus for work in the reporting period has been to define protocols and analyses through a Science Verification Test (SVT) and Experiment Verification Test (EVT). The SVT was completed in June of 2022 and EVT in October 2022. Both tests were successful with a rating of excellent across all of the experiment’s success criteria.

Overview of procedures: The combination of SVT and EVT have allowed definition of experiment procedures for the flight experiment. Briefly, seeds, Trichoderma spores, or seeds inoculated with Trichoderma spores will be planted on 12 cm square Petri dishes containing gel growth medium supplemented with ½ strength Linsmaier and Skoog salts. A polyester mesh (1 mm mesh size) supported by a custom rim 3d printed in polyethylene tetraphthalate will be laid on the gel surface prior to planting. This aids in subsequent harvest of plants and fungal hyphae as the crew can lift off the mesh with all samples attached and intact for further processing. After planting, germination will be delayed for pre-flight operations by holding the plates at 4 °C. The samples will then be installed in the Veggie growth chamber and grown for 14 days with photography every other day.

At 14 days, the tomato seedling roots are close to the bottom of the plate, and this growth feature defines when harvest will occur. The plates are then opened and mesh with samples attached is removed, wrapped in foil, and snap frozen between aluminum bricks conditioned to -160 °C. These samples are then stored at -80 °C until analysis. Current work is aimed to modify the harvest procedures to allow them to operate inside the Life Science Glovebox. This approach is to provide an extra level of containment on orbit.

It is important to delay germination of both the tomato seeds and the Trichoderma spores before insertion into the Veggie on orbit. Testing has revealed that germination of both the tomato and Trichoderma is delayed by storage in the dark at 4 °C. This approach proves to be effective for 2 weeks, at which point the Trichoderma spores begin to germinate and grow.

Once installed in the Veggie at room temperature, the seeds and Trichoderma geminate and grow until the plants fill the 12 cm Petri plate growth space after 14 days. At this point, the plants are well developed with true leaves and highly branched root systems and the Trichoderma colonies have grown to cover ~ 50% of the growth area. Analysis of the images through the growth time course shows that inoculation with Trichoderma alters both growth of the primary root and branching patterns, as predicted from previous research on this interaction.

Biochemical Assays: In addition to growth analysis from the imaging described above, the frozen samples have been successfully assayed for chlorophyll and carotenoid content (as a measure of photosynthetic capacity), accumulation of anthocyanin (as a general stress marker) and levels of malondialdehyde, (as a biochemical marker for oxidative stress). Testing of all of these assays has been highly successful in both the SVT and EVT.

RNA quality and quantity: One major aim of this research is to perform transcriptional profiling of the samples using RNA-seq. To test how well the samples perform for these kinds of assays, quantitative polymerase chain reaction (qPCR) has been used as a proxy for full RNA-seq analyses. Frozen samples provided material for isolation of RNA of sufficient quantity and quality (average A260/280 of 2.00) to separate root and shoot samples (average of 3.6 µg RNA/root or shoot sample/plate) and analyze them for quantitative PCR. Three representative genes were assessed in SVT and EVT as proof-of-concept that the nucleic acid will be suitable for broad-scale molecular analyses: PRP1b is a pathogenesis-related protein that is known to be induced in response to successful Trichoderma infection of the plant roots, HSP22 is a heat shock protein that is a marker for oxidative stress responses, and NT is a nitrate transporter that should be unresponsive to Trichoderma infection. The reference gene for the qPCR analysis was Elongation Factor 1-alpha. These genes were successfully analyzed and preliminary analyses show significant elevation of PRP1b and HSP22 in plants treated with Trichoderma, whereas NT was unaffected.

Stable C-isotope analysis: In order to monitor photosynthetic parameters such as water use efficiency, stable isotope analysis is also planned to be used. To test the sampling method for these analyses, 4 gas samples were taken of the ambient atmosphere in the vicinity of the Veggie using Silonite MiniCans, with 2 samples at the start (day 0) of the experiment and 2 at the end (day 14). These samples were shipped to the University of New Mexico for analysis by the Hanson lab. All 4 samples were successfully analyzed, showing an average 12C:13C CO2 isotopic ratio of 25.6 +/- 4.6 per mil. These provide the baseline atmospheric measurements for use in modeling from tissue stable isotope content.

The major focus for work in the reporting period has been to optimize and define flight procedures and protocols. Extensive testing revealed that 12 cm square Petri dishes offered the required balance between plant and fungal growth. Harvesting of both tomato and fungal samples has also been optimized. The plants/fungus are grown on the surface of a nutrient gel. However, removing the plant and associated fungal hyphal mat intact was initially challenging, as the fungus adhered to the growth matrix. A series of surface substrates was therefore tested before settling on 1 mm pore size cotton fiber mesh, which allowed both normal plant growth and the lifting of the whole plant and fungal samples from the gel surface intact. The large pore size of this mesh likely provides enough area of direct contact between the plant roots and the Phytagel growth medium below the cloth to sustain "normal" plant growth. The mesh was laid on the nutrient gel (1/2 strength LS medium with 1% Phytagel) surface and then seeds were placed on this layer and the plants grown along the surface. This approach allowed the whole plant, with Trichoderma attached, to be harvested by peeling the cotton substrate off of the surface of the gel. These samples were then snap frozen by placing the entire sample in a foil bag and rapidly freezing using aluminum blocks conditioned to -160°C.

Delaying germination: Cold temperatures were optimized to delay germination of both the tomato seeds and the Trichoderma spores before insertion into the NASA Vegetable Production System (Veggie) in orbit. Germination of both the tomato and Trichoderma is delayed by storage in the dark at 4°C for 2 weeks. At this time point, the Trichoderma (but not the tomato seeds) begins to germinate and grow, setting the limit on the timing of storage prior to initializing the experiment in the Veggie.

Confirmation of fungal inoculant: Sequencing data from samples of the fungal isolates to be used from 2 diagnostic genes (ITS1/2 and TEF1) was BLASTed against both the National Institutes of Health (NIH) sequence databases and using the species identification tools at Trichokey.com used to confirm the fungal inoculant as T. harzianum.

RNA quality and quantity: Initial testing indicates a single 3-week-old tomato seedling from each plate will provide > 1µg of RNA of sufficient quality to analyze the required separate root and shoot samples by both RNA-seq and quantitative (polymerase chain reaction) PCR tests.

II. GROUND-BASED CHARACTERIZATION

In parallel with the efforts to define flight procedures, the interaction between T. harzianum and both tomato and Arabidopsis plants has been further characterized. A key observation is that, as proposed by several groups, the growth promoting factor of the interaction between plant and fungus seems to be delivered by volatiles from the fungus. Thus, increased growth and stress resilience in the plants have been evident when the Trichoderma is confined away from the plant using a spit plate system (i.e., plates with a divider separating fungal growth media from the plants). Unexpectedly, growth of the plant roots has been seen towards the fungus and fungus towards the plants, suggesting that the plant root may also be releasing volatiles that are detected by the fungus. Defining the potential mechanism of both of these directional growth responses is a key goal for the coming year as, at a practical level, it may be possible to grow the fungus remotely from direct contact with the plant and/or just deliver the fungal volatiles but still gain the beneficial effects on plant growth.

III. PRESENTATIONS AND OUTREACH

These spaceflight-related projects have been presented at multiple outreach events at venues -- ranging from colleges and universities such as Elizabethtown College, Oregon State University, and the Instituto de Biologia Experimental e Tecnologica in Oeiras, Lisbon, Portugal -- to outreach to the general public at events such as the University of Wisconsin’s Science Expeditions, the Friends of Allen Centennial Garden and Madison Master Gardeners, and the Sidmouth Science Festival in the UK, as well as to school students as part of "Space Camp" at the Deke Slayton Memorial Space and Bike Museum in Sparta, WI.

Specifically, we propose to:

(1) Analyze the kinetics of growth and the transcriptome, ionome, and photosynthetic responses of tomato plants to spaceflight.

(2) Compare these responses in plants growing with or without the beneficial root symbiotic microbe Trichoderma harzianum.

(3) Use comparisons to ground-based control experiments, clinostats, and random positioning machines to ask whether such changes are likely linked to the microgravity element of spaceflight and how far these ground-based microgravity analogs really do trigger responses seen in the spaceflight environment.